Discovery Proteomics and Absolute Protein Quantification Can Be Performed Simultaneously on an Orbitrap-Based Mass Spectrometer

Abstract

Mass spectrometry (MS) has steadily moved into the forefront of quantification-centered protein research. Protein cleavage isotope dilution MS is a proven way for quantifying proteins by using an isotope-labeled analogue of a peptide fragment of the parent protein as an internal standard. Parallel reaction monitoring (PRM) has become the go-to approach for such quantification on an Orbitrap-based instrument as it is assumed that the instrument sensitivity is enhanced. We performed a comparative study on data-dependent acquisition (DDA) and PRM-based workflows to quantify egg yolk protein precursors or vitellogenins (VTGs) Aa, Ab, and C in striped bass (Morone saxatilis). VTG proportions serve as a developmental measure of egg quality, possibly changing with the environment, and have been studied as an indicator of the health of North Carolina stocks. Based on single-factor analysis of variance comparisons of mean VTG amounts across fish from the same sample groupings, our results indicate that there is no statistical difference between MS1-based and MS2-based VTG quantification. We further conclude that DDA is able to deliver both discovery data and absolute quantification data in the same experiment.

Figure 1

Absolute quantification of VTG Aa in striped bass: DDA and PRM analyses.

Figure 2

DDA-MS1—TIC with extracted ion chromatograms for [M + 2H]²⁺ NAT and SIL peptides from target VTGs Aa, Ab, and C. The ovarian biopsy sample came from striped bass no. 52 from the Roanoke River. The DDA nano-LC–MS/MS run no. 3 is shown here.

Figure 3

PRM (PRM-MS1)—TIC with extracted ion chromatograms for [M + 2H]²⁺ NAT and SIL peptides from target VTGs Aa, Ab, and C. The ovarian biopsy sample came from striped bass no. 52 from the Roanoke River. The PRM nano-LC–MS/MS run no. 3 is shown here.

Figure 4

Spacing of MS1 events in PRM-MS1 and DDA-MS1 analyses for doubly charged [M + 2H]²⁺ peptide TEGLQEALLK from VTG Aa. The ovarian biopsy sample came from striped bass no. 52 from the Roanoke River. The PRM and DDA nano-LC–MS/MS run no. 3 is shown here.

Figure 5

Spacing of MS1 events in PRM-MS1 and DDA-MS1 analyses for doubly charged [M + 2H]²⁺ natural peptide IATALVDTFAVAR from VTG Ab. The ovarian biopsy sample came from striped bass no. 52 from the Roanoke River. The PRM and DDA nano-LC–MS/MS run no. 3 is shown here.

Figure 6

Spacing of MS1 events in PRM-MS1 and DDA-MS1 analyses for doubly charged [M + 2H]²⁺ natural peptide YFQATTLGLPLEISK from VTG C. The ovarian biopsy sample came from striped bass no. 52 from the Roanoke River. The PRM and DDA nano-LC–MS/MS run no. 3 is shown here.

Figure 7

SIL peptide peak area ratio PRM-y8/DDA-MS1. This ratio shows the relative reduction in the number of ions actually detected for the y-8 ion vs the precursor ion. There is not a 1-to-1 precursor-to-product ion conversion in the fragmentation process in any mass spectrometer.

Figure 8

SIL peptide precursor ion abundance (peak height) ratio PRM-y8/DDA-MS1. This ratio shows the relative reduction in the number of ions actually detected for the y-8 ion vs the precursor ion. There is not a 1-to-1 precursor-to-product ion conversion in the fragmentation process in any mass spectrometer.